M13 Tailed Primer Protocol
Primer Design
“M13” labeled sequences:
FAM = TTT CCC AGT CAC GAC GTT G
NED = TAA AAC GAC GGC CAG TGC
VIC = GCG GAT AAC AAT TTC ACA CAG G
Identical sequences appended to the 5’ end of the locus-specific forward primers.
In addition, the following sequence is appended to the 5’ end of the reverse primer:
+A/-A Tail = GTTTCTT
This sequence serves to drive the non-templated addition by Taq of a base (usually +A)
onto the 3’ end of the labeled strand. This allows for easier allele scoring (by eliminating
the –A peak).
Primer Mixes
Labeled primer mix stock
52 µls of 500 uM M13, dye-labeled oligos (Applied Biosystems) added to 50 mls of 10
mM Tris
 each of the three labeled M13 primers at 0.52 uM in primer mix stock
Add the following to 2990 uls of M13 primer mix stock:
7.7 µls of each 100 uM Forward (usually a 40-mer; contains the M13 leader sequence)
x 3 (multiplex 3-deep)
77 µls of each 100 uM Reverse (usually a 26-mer; contains the +A leader sequence)
x 3 (multiplex 3-deep)
plus 1016 µls of 10 mM Tris to bring to volume
= 4260 µls of reagent; enough for 8 microtiter plates of PCR
 Forward/ M13 /Reverse/ are in final ratio of 0.1x/0.2x/1.0x
 X = 1.8 uM